Abstract

A 'better' Escherichia coli K-12 genome has recently been engineered in which about 15% of the genome has been
removed by planned deletions. Comparison with related bacterial genomes that have
undergone a natural reduction in size suggests that there is plenty of scope for yet
more deletions.

Minireview

Why should one want to design a better bacterium? One answer is that this is one way
of really testing our understanding of how a living cell works - by making predictions,
manipulating the object, and seeing what we get. This is the province of synthetic
biology, whose ultimate goal is to understand life by constructing it from scratch;
it is hoped that along the way will emerge an understanding of the properties of living
cells and organisms that is difficult to arrive at by conventional investigation of
the organisms themselves [1,2]. Much progress has been made recently towards designing and synthesizing novel biological
organisms from a set of standardized parts [3], such as protein-coding genes, regulators, and terminators, as listed on the BioBrick
website [4].

In contrast, in work recently published in Science, Posfai et al. [5] have taken a 'deconstructionist' approach to redesigning life. Specific regions of
the Escherichia coli K-12 genome were targeted for deletion with the intention of improving the production
potential of this model organism. As an unanticipated side effect, they have come
up with a bacterium that is even better than the parental strain for some purposes,
in that it is more efficiently electroporated and accurately propagates unstable recombinant
genes and plasmids. It is interesting to compare these engineered reduced genomes
[5] with the genomes of other bacteria within the Enterobacteriaceae, some of which are
endosymbionts whose genomes have become dramatically reduced during evolution.

Smaller is indeed often better, as people who travel frequently or who worry about
buying fuel for their cars know. Posfai et al. [5] chose which genes and genomic regions to remove on the basis of several criteria,
including "troublesome sequences" such as insertion sequence (IS) sites and transposable
elements that appear to code only for their own replication, and repeat regions that
can cause homologous recombination. They also removed some regions that are not present
in all E. coli genomes, and so are unlikely to be essential for basic properties such as growth.
There are many large regions throughout the E. coli K-12 genome that are not conserved among other E. coli genomes, but given the variation in genome size between different strains, with differences
of more than 1 million base pairs (20% of the genome) being common, this is perhaps
not surprising.

To make the deletions, synthetic oligomers containing regions homologous to target
sites flanking the desired region were used. Regions were deleted by recombination
mediated by the phage lambda Red system, and done in a way that gave 'scarless' deletions
where no marker sequence was left. The strains with deletions were then tested for
growth in minimal media. Finally, as one last step to check for quality, the reduced
strains were hybridized to tiling microarrays of the E. coli K-12 genome. The first reduced strain yielded surprising results. In the words of
the authors: "Alarmingly, we found five unexpected copies of IS that had transposed
to new locations since the project began in 2002." Thus new strains were made, which
were shown to be free of IS elements. The engineered strains had similar growth rates
to their parent strain, and the electroporation efficiency of engineered strain MDS42
was 100 times greater than for the original E. coli MG1655 K-12. Furthermore, plasmid genes that were unstable in MG1655 were found to
be completely stable in the engineered strains. IS mutagenesis is a natural defense
against deleterious genes, and is normally helpful to bacteria in the wild, but is
detrimental when one wishes to grow these genes in laboratory strains of E. coli.

Natural genome reduction

If synthetic biology can be used to design a reduced E. coli genome with some desirable new functions, what about 'non-synthetic' biology - that
is, evolution? Is there anything that we can learn from evolutionary biology about
how to make a reduced E. coli genome? Soon after Posfai et al. [5] published their paper, a study by Wu et al. [6] appeared on the reduced genomes of two very different bacteria living inside an insect
called the glassy-winged sharpshooter (Homalodisca coagulata) [6]. One of these bacteria (Baumannia cicadellinicola) belongs phylogenetically to the same group as E. coli, which inspired me to make a comparison of all of the engineered E. coli genomes of Posfai et al. [5] with other enteric bacterial genomes sequenced so far (Table 1).

Table 1. List of currently sequenced genomes from the family Enterobacteriaceae of the γ-Proteobacteria

The B. cicadellinicola genome is towards the bottom of the table, but there are four known genomes in this
family that encode an even smaller number of proteins. The genome at the bottom of
the list (Buchnera aphidicola strain BBp) codes for only 504 proteins, or less than 10% of the number of proteins
encoded by the larger E. coli genomes (5,379 proteins in E. coli CFT073). The smallest 'normal', free-living enterobacter (apart from the newly engineered
E. coli genomes) is a Yersinia pestis strain (Mediaevalis), with 3,895 genes, or only 72% of the number of genes found in
the largest enterobacterial genome. Furthermore, only slightly more than half of the
Y. pestis Mediaevalis genes have homologs in the CFT073 genome (52% - that is, 2,938 Y. pestis genes/5,379 E. coli CFT073 genes). Thus, just on the basis of gene diversity within the enteric bacteria,
it seems that perhaps half or more of the genes in the larger enterobacterial genomes
might be expendable - at least under laboratory growth conditions. Indeed, only 620
E. coli K-12 genes have been found experimentally to be essential for growth in rich media,
while 3,126 genes were found to be dispensable for growth under this condition [7]. This indicates that there could well be more room for engineered deletions in the
E. coli genomes.

Figure 1 compares the proteins encoded by some of the genomes in Table 1. E. coli K-12 isolate MG1655 was the genome used as a starting point by Posfai et al. [5] to make the reduced genomes, and it is used as the reference here. The locations
of all the deletions engineered by Posfai et al. [5] are indicated outside the circles. The location of each gene from the E. coli MG1655 genome is used to visualize the best hit based on a BLAST search of all the
proteins encoded by the different genomes indicated in the legend. A strong hit is
represented by a solid bar so the extensive solid regions in the circles represent
regions of homology with the other genomes. Each circle represents a different genome,
with the outer five circles representing nonreduced genomes, and the inner three circles
reduced genomes. Thus, the outermost circle is for the Y. pestis genome, followed by a Salmonella genome, and then three E. coli genomes. Note that many of the planned deletions made by Posfai et al. [5] correspond to gaps in the otherwise mostly solid dark circles; these gaps represent
large chromosomal regions lacking homology in terms of protein sequences in the other
genomes.

Figure 1. A BLAST atlas diagram of eight enteric bacterial genomes, compared to the reference
E. coli K-12 isolate MG1655. The outer five circles are other similar genomes from E. coli, S. enterica and Y. pestis (outer circle), while the inner three circles reflect the reduced genomes (from the
innermost circle outwards) of B. aphidicola, B. cicadellinicola and S. glossinidius. Each colored circle represents the BLAST score of the best hit of the given bacterial
proteome versus the gene at a given location in the reference E. coli MG1655 genome. Note that the scale is -log E-value, which means that the strongly
colored regions have an E-value of less than 10-100, which corresponds to a very good match. The locations of all the deletions engineered
by Posfai et al. [5] are indicated outside the circles. Coding sequences of the reference genome
(E. coli k-12 strain MG1655) are indicated as blue and red blocks, corresponding to genes orientated
clockwise or counterclockwise. The gaps in the E. coli W3110 genome are due to rRNA operons and other non-coding RNAs, which do not show
up on the protein BLAST results.

The reduced genomes are shown in the inner circles. B. aphidicola strain BBp is the smallest genome, and is depicted as the orange inner circle, which
has few hits, as expected, as this genome encodes so few proteins. The B. cicadellinicola genome is the next circle (red), and the third is Sodalis glossinidius, which is a genome that is undergoing reduction, but still contains about 2,500 genes,
as well as about 1,000 pseudogenes [8]. This circle contains more hits, although it is still a bit sparse compared to the
inner three circles, which have large regions where nearly all of the proteins are
conserved.

These reduced genomes contain only about 10% of the genes in the larger E. coli genomes from which they originated long ago. This raises many questions. What about
the remaining 90%? Does E. coli really not need most of these genes? Some are certainly redundant - a necessary condition
for robust systems [9] - and the definition of 'essential genes' might include some genes that do not give
a lethal phenotype when deleted [10].

Is it possible to model which genes would remain, and which 90% or so could be removed,
under the right conditions? A model of E. coli metabolism was recently used to generate reduced genomes in silico [11], and to compare these genomes with the endosymbiotic genomes shown at the bottom
of Table 1. The idea was to use a known metabolic environment and then to model random gene
loss, and evaluate relative viability. If the gene loss had no apparent effect, then
another gene would be removed, and this process was repeated until a minimal genome
was obtained.

Two different endosymbiotic bacterial environments were modeled - those of Buchnera and Wiggelsworthia - and the model predicted the gene content of the two genomes to about 80% accuracy
[11].

There are, of course, several different ways to arrive at the same reduced genome,
but by looking at which genes are necessary to perform core metabolic activities (for
a given endosymbiotic environment, it should be stressed), it is possible in general
to predict the genes that are likely to remain in a reduced genome. This information
can then be used in future experiments to design better genomes, tailor-made for specific
applications. Posfai et al. [5] were not intending to manufacture a 'minimal genome' such as the highly reduced ones
discussed here, but rather they simply wanted to engineer an E. coli genome that would be a better 'workhorse' - that is, it would be easier to get DNA
into the cells, and the DNA and its gene products would be stable once it was there.
There are others, however, who do have the aim of using synthetic biology to design
and manufacture a minimal genome [12]. Perhaps the time is near when mircobiology will join the engineering sciences.

Acknowledgements

I would like to thank Michael Sismour for useful comments about synthetic biology
and the Danish Center for Scientific Computing for funding.